We have constructed and identified cDNA classes of human parainfluenza virus type 3 (PI3 virus) mRNAs encoding the viral hemagglutinin-neuraminidase (HN) glycoprotein, nucleocapsid phosphoprotein (P), major nucleocapsid (N) protein, matrix (M) protein, and fusion (F) glycoprotein. Northern blot hybridization identified the corresponding major viral mRNAs extracted from PI3 virus-infected cells. cDNAs of the N, P and M mRNAs were identified by hybrid-selected translation and hybrid-arrest of translation in vitro. A cDNA clone containing the complete HN mRNA was sequenced, and the identification of this cDNA was confirmed by comparison of the predicted protein sequence with a partial sequence obtained by direct amino acid sequencing of authenic HN protein purified from infected cells. Based on partial nucleotide sequence information for the N, P, M and F cDNAs, oligodeoxynucleotides were synthesized and used to direct dideoxynucleotide sequencing of purified viral genomic RNA. To date, this analysis determined a partial order of genes on the PI3 genone: 3' N-P-M-(F)-HN-5'. Further sequence analysis of cDNAs and viral genomic RNA will provide a complete enumeration of viral genes and a complete determination of viral gene sequences.